4.4.3. Group III intron :
Group III auto splicing are rich in A U and ranging from 95 to 110 nucleotides. Group III introns are shorter than group I and II intron but show similarity with group II introns, having conserved sequences proximal to the splicing site specially 5’ splice site. Splicing occurs through lariat and circular RNA formation and identified in chloroplasts of euglenoid protists mRNA genes.
4.5. Homing and Retrohoming process :
In homing and retrohoming mechanism an intron is insert a within an intron less cognate sequence, which consist of a particular specific site. Some intron of group I and group II has ORF. These ORF translated in enzyme maturase, endonuclease and reverse transcriptase.
Homing intron posses two enzyme maturase and endonuclease. Maturase enzyme help in splicing while endonuclease responsible for homing.
A specific restriction site present on DNA sequence gene which not possess homing intron, but this DNA sequence (restriction site) is present only in copy of target gene. In a particular cell, two copies must be present for homing. Thus intron coding endonuclease makes introns as mobile element.
In retrohoming intron, maturase, endonuclease and reverse transcriptase genes are found. Endonuclease cut the intron by double strand break and reverse transcriptase make DNA copy of this mRNA, and thus insert this DNA copy into targeted gene, thus move through RNA arbitrated (mediated) mechanism.
4.6. Twintrons :
Introns-within-introns called twintrons. This discovered in Euglena chloroplast genome as a group II intron within another group II intron and excised by sequential splicing reactions by lariat intermediates with in internal and external introns.
4.7. Alternative Splicing:
Alternative splicing is a process in which exons are joined together in differential method and make different polypeptide from a single mRNA transcript. In alternative splicing introns can be retained or exons can be skipped or extended.
One of the best example of alternative splicing is sex determination in Drosophila. In Drosophila sex of fly is determine by ratio of X-chromosome to autosomes. Fly with X/A ration = 1 develope into female and If ratio of X/A is 0.5 develop into male. There are three gene have an important role to transmit this information to other cell in turn upto the genes whose product involve in development of male and female characterstic. They are sxl (sex lethal), tra (transformer) and dsx (double sex). These gene are transcribe seperatly but their splicing is regulated by product of these gene in sequential cascade manner.
Product of sxl gene help in splicling of tra gene and product of tra gene help in splicing of dsx gene. All genes are normal function fly become female. If one of the gene form dysfunction product fly become male. In this Splicing mechanism sxl product act as repressor and block the 3’splice site thus functional tra protein responsible for female form by the loss of two exon, and this tra protein responsible for the formation of female specific dsx splice mRNA and so female specific dsx protein containing 430 amino acid and in this case tra act as activator.
If sxl protein dysfunctional than male specific tra protein form and in turn male specific dsx protein formed containing 550 amino acid.
Another example of trans splicing from muscle protein of mammals Troponin T. The Troponin T gene transcript contain five exon. When during splicing the fourth exon skipped than spliced mRNA form α troponin T. and when during splicing the third exon skipped than spliced mRNA form β troponin T.
In some lower organism like trypanosoma special type of splicing process found in which exon of two different transcripts ligated together. In trypanosomes, a leader sequence present at the 5’end of m-RNA. This leader sequence joined to other m-RNA exon by trans-splicing. ‘Y’ shaped intron exon intermediate is form This Y-shaped intermediate is analogous to lariat. Now leader exon attacks the splice site which is located at branched intron and coding exon. Thus 2 type structure are obtain one is mature mRNA and second is Y-shaped intron.
Trypanosomas cause african sleeping sickness, till date there is no cure of this disease because trypanosomas has ability to change their receptor, here, N terminal sequence where the Ab bind is continuously change due to trans-splicing and the c terminal remain constant. Trans-splicing also found in C. elegans.
4.9. RNA Editing
RNA editing is a process in which RNA transcript has some different bases from it template DNA bases. That means in RNA editing process either some bases are added after transcription or some bases are removed from transcript. Somewhere instead of addition or deletion, some base replace with another bases.
Thus RNA editing occur by two mechanism
2. Addition or deletion
1. Substitution editing: Substitution editing occurs due to chemical alteration in individual base. This alteration cause by some enzymes. These enzymes change one nucleotide into another nucleotide.
For example adenosine deaminase acting on RNA (ADARS) converts Adenine (A) to I (inosine) known as A-I editing. I (inosine) behave like Guanosine in DNA and ribosome also translates Inosine as a Guanosine. Cytidinedeaminase convert Cytocine to Uracil known as C- U editing.
Human APO-B gene is best example of substitution editing. This gene expressed in liver cell and intestinal cell of human. APO-B contains 29 exon which are separated by 28 Intron. In 29 exon total 4564 codons are present. At codon 2153 (CAA) code for glutamine amino acid.
In human liver cells, this get transcribed and translated normally and gene product called apolipoprotein B –100 (contain 4563 amino acid) apolipoprotein B 100 work as cholesterol and lipid transport in blood. But in intestinal cell RNA editing occur in transcript of APO-B gene and at 2153 codon cytocinedeaminase convert Cytocine to Uracil. Thus codon CAA changes in UAA which is stop codon. Due to this final protein contains only 2152 amino acid rather than 4563. In intestine this protein known as APO-48 and help in cholesterol absorption from dietary lipids.
Substitution editing also found in tRNA, rRNA of chloroplast and mitochondria of plants, Receptor of neurotransmetter in mammals brain, encoding mRNA also processed through editing process.
Insertion and deletion editing :
Insertion and deletion process requires RNA molecules known as guide RNA (gRNA). gRNA have complementary sequence to editing site within its central domain, which is in between 5’ (having complementary sequence to pre-mRNA) and 3’ (having poly U tail) end. First step in addition and deletion process requires binding of guide RNA with unedited transcript.
If some mismatch bases are present in transcript, get loop out, endonucleolytic cut makes and remove mismatched bases. Now another enzymes terminal uridylyl transferase (TUT) add uridine in removal site. Editing occur in 5’-3’ direction. All enzymes responsible for cutting, insertion, deletion and ligation present in 20 S editosome. RNA editing by gRNA reported in kinetoplastid protists mitochondrial mRNAs.
Mechanism involve in deletion editing:
- U deletion: A nuclease (endonuclease-because only endonuclease break phosphodiester bond) cut RNA from 3' site of U, which to be deleted.
- Now uridyl exonuclease remove UMP at the end of the left-hand RNA fragment, base pairing occurs between bases of the hn mRNA and base of gRNA.
- RNA ligase join the two fragment of pre mRNA together.
Insertion editing :
- First guide RNA bind with mRNA. If guide RNA fail to exact complementary binding than phosphodiester bond break by nuclease and free 3’OH get created.
- Now Terminal Uridylyl transferase (TUTase) insert U base in mRNA.
- Finally RNA ligase join two fragments.
RNA used as catalysts in living cells, other than their known roles in information storage and as molecular architectural frameworks are called ribozymes. This idea was so profoundly contrary to the central dogma of molecular biology that it resulted in the award of a Nobel Prize to two of the early proponents, Thomas Cech and Sidney Altman. The discovery was twofold – RNA segments that cut themselves out of larger RNAs (self-splicing introns) and a protein-assisted RNA enzyme (ribonuclease P) that cuts the leader sequences off all transfer RNAs throughout the three organismal domains. RNAs with catalytic activity, revealed the extraordinary characteristic of this molecule, and collaborated the idea that RNA was the first informative polymer. The catalytic properties of ribozymes are majorly due to the capacity of RNA molecules to fold into particular structures. The versatile structures of RNA can allow a single RNA sequence to fold in more than one structure providing them with the virtue of multiple functions.
Ribozymes are found in nucleus, mitochondria and chloroplasts of eukaryotes, as well as in some viruses. These are grouped by their chemical type, but regardless of the type, all RNA are associated with metal ions such as potassium (K+) or magnesium (Mg2+), which play essential roles in catalyzing reactions.
Most ribozymes are involved in the processing of RNA. They either serve as “molecular scissors” or as “staplers” that cleave and ligate molecules of RNA. Although most targets of ribozymes are RNA, evidence suggests that the assembly of amino acids into a protein that occurs during translation is also catalyzed by RNA, meaning the ribosomal RNA is also a ribozyme.
Group II introns are autocatalytic ribozymes that catalyze RNA splicing and retrotransposition. Splicing by group II introns plays an essential role in the metabolism of plants, fungi, and yeast and contributes to genetic variation in many bacteria. Group II introns are also responsible for genome evolution. The structure and catalytic mechanism of group II introns have been elucidated through a combination of genetics, chemical biology, biochemistry, and crystallography. A central active site, containing a reactive metal ion, catalyzes both steps of self-splicing. These studies provide insights into RNA structure, folding, and catalysis, as they raise new questions for the behavior of RNA machines.